An experimental system will be developed to provide direct evidence concerning (a) the pathway of intracellular enzyme degradation, including the existence of intermediates and subcellualr (cytoplasmic or lysocomal) localization of same; (b) the role of coenzyme dissociation in initiation of enzyme degradation; and (c) specific structural features which contribute to degradation of different enzymes. Enzymes will be labeled in vivo, purified, and the native purified labeled protein put back into cell by microinjection. These injected, labeled proteins will then serve in vivo as specific labeled substrates for intracellular enzyme degradation. Disappearance of this label in new polypeptide species, i.e. intermediates, will allow delineation of the pathway. In vitro modification of labeled proteins, including removal of coenzyme, prior to their injection will be used to examined structural features which contribute to enzyme degradation. This represents a new approach to the study of intracellular protein turnover. Enzymes chosen for initial study are ornithine decarboxylase and tyrosine aminotransferase; both are dependent on the coenzyme, pyridoxal 5'-phosphate. Ornithine decarboxylase, which has the shortest in vivo half-life known, is the rate-limiting enzyme in polyamine biosynthesis and has been used as an indicator of tumor promotion. Tyrosine aminotransferase, which also has a short half-life, is known to respond to hormonal control. Initial injection experiments will use the slime mold, Physarum polycephalum, because of its large size.